Features of influenza HA required for apical sorting differ from those required for association with DRMs or MAL.

The influenza virus hemagglutinin (HA) is sorted to the apical membrane in polarized epithelial cells and associates with detergent-resistant membranes (DRMs). By systematic mutagenesis of the transmembrane residues, we show that hemagglutinin requires 10 contiguous transmembrane amino acids to enter detergent-resistant membranes and that the surface of the trimeric hemagglutinin transmembrane domain facing the lipid environment as well as that facing the interior of the trimer is important for stable association with detergent-resistant membranes. However, association with detergent-resistant membranes was not required for apical sorting. MAL/VIP17 is a protein that is required for apical transport and a small fraction of hemagglutinin co-precipitates with MAL. Mutations that prevented HA from being isolated in detergent-resistant membranes decreased co-precipitation with MAL. The hemagglutinin and MAL that co-precipitated were contained in a detergent-resistant vesicle. However, most of the co-precipitation of newly synthesized hemagglutinin with MAL occurred only after the majority of hemagglutinin reached the cell surface. Both the timing and the limited extent of co-precipitation suggest that the majority of vesicles containing hemagglutinin and MAL are not the detergent-resistant membrane transport intermediates carrying hemagglutinin from the TGN to the apical surface.